Methicillin-resistant Staphylococcus aureus (MRSA) is an important infectious human pathogen responsible for diseases ranging from skin and soft tissue infections to life threatening diseases. Emerging research has demonstrated that MRSA infections have a significant clinical impact on individuals with chronic underlying diseases such as cystic fibrosis (CF), diabetes and osteomyelitis in where antibiotic pressure and metabolic adaptions may favor the adaptability of S.aureus to establish long persistence. Ceftaroline (CPT) is the only FDA-approved cephalosporin targeting PBP2a with strong antibacterial activity including MRSA. We have examined the molecular basis of both CPT-high(CPT-HR) and -intermediate (CPT-IR) resistant MRSA strains isolated for the first time in the USA displaying MICs to CPT of 2-8 ?g/ml and ?32 ?g/ml, respectively. Although mutations in the mecA gene have been associated to CPT resistance, the molecular mechanisms have not been fully elucidated. Our data suggest that the CPT-HR strain harbored three SNPs, two of them in the penicillin-binding pocket of PBP2a that result in replacement of amino acids directly acting as gatekeeper to the transpeptidase active site, while the third mutation localizes at the non-penicillin binding or dimerization domain. Related tochanges in mecA, non-synonymous mutations were also identified in pbp2. In addition, other relevant nonsynonymous SNPs were present in thyA (gene coding for the thymidylate synthase) and mutS and mutL (DNA mismatch repair) genes. Moreover, we determined that CPT-IR strains displayed a very significant increase in CPT-induced mecA expression that, accompanied with mutations in PBP2, together may interfere with CPT's ability to repress completely both PBP2a and PBP2a/PBP2 interactions. A common feature consistently found in CPT-IR and -HR strains was their high level of resistance to imipenem (IPM).This proposal intends to elucidate the molecular bases of CPT-HR and CPT-IR in MRSA clinical strains, and to provide new therapeutic antimicrobial combinations for the treatment of CPT-resistant MRSA infections. The Specific Aims of this proposal are: Specific Aim#1 To investigate the mechanistic significance of PBP2 perturbations in CPT-resistance Specific Aim #2 To define S. aureus metabolic adaptations linked to thyA mutations during development of CPT-resistance. Specific Aim #3 To identify the genetic determinates and analyze the role of carbapenems in the acquisition of CPT-resistance in clinical MRSA strains.